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Biomedical subjects

M Fujishiro

Publications and source records attributed to M Fujishiro.

At least 37 records · Page 2Linked to original sources
PubMed2001

Palmitoylation of the canine histamine H2 receptor occurs at Cys(305) and is important for cell surface targeting.

To determine the presence and functional role of the histamine H2 receptor (H2R) palmitoylation, a receptor with a Cys(305) to Ala (A(305) receptor) mutation was generated. Wild-type (WT) and A(305) receptors were tagged at their N-termini with a hemagglutinin (HA) epitope. WT, but not A(305), receptors incorporated [3H]palmitate by metabolic labeling, indicating that the H2R is palmitoylated at Cys(305). Immunocytochemistry of WT and A(305) receptors expressed in COS7 cells revealed WT receptors to be distributed at the plasma membrane, while the majority of A(305) receptors were localized intracellularly with only a small portion being at the plasma membrane. However, the affinity of the A(305) receptor for tiotidine was comparable to that of the WT receptor. In addition, when the amounts of cell surface receptors as determined by anti-HA antibody binding were equivalent, A(305) receptors mediated production of more cAMP than WT receptors. Preincubation of COS7 cells expressing each receptor with 10(-5) M histamine for 30 min reduced subsequent cAMP production in response to histamine via the receptors to similar extents, indicating that palmitoylation is not necessary for desensitization. In addition, cell surface A(305) receptors were capable of being internalized from the cell surface at a rate and extent similar to those of WT receptors. Finally, CHO cell lines stably expressing either WT or A(305) receptors were incubated with 10(-5) M histamine for 1, 6, 12 and 24 h. Total amounts of WT and A(305) receptors, as determined by tiotidine binding, were reduced by incubation, indicating downregulation. Downregulation of the A(305) receptor was more extensive than that of the WT receptor. Thus, palmitoylation of the H2R might be important for targeting to the cell surface and stability.

Adenylyl Cyclases↗
PubMed2001

MKK6/3 and p38 MAPK pathway activation is not necessary for insulin-induced glucose uptake but regulates glucose transporter expression.

p38 mitogen-activated protein kinase (MAPK), which is situated downstream of MAPK kinase (MKK) 6 and MKK3, is activated by mitogenic or stress-inducing stimuli, as well as by insulin. To clarify the role of the MKK6/3-p38 MAPK pathway in the regulation of glucose transport, dominant negative p38 MAPK and MKK6 mutants and constitutively active MKK6 and MKK3 mutants were overexpressed in 3T3-L1 adipocytes and L6 myotubes using an adenovirus-mediated transfection procedure. Constitutively active MKK6/3 mutants up-regulated GLUT1 expression and down-regulated GLUT4 expression, thereby significantly increasing basal glucose transport but diminishing transport induced by insulin. Similar effects were elicited by chronic (24 h) exposure to tumor necrosis factor alpha, interleukin-1beta, or 200 mm sorbitol, all activate the MKK6/3-p38 MAPK pathway. SB203580, a specific p38 MAPK inhibitor, attenuated these effects, further confirming that both MMK6 and MMK3 act via p38 MAPK, whereas they had no effect on the increase in glucose transport induced by a constitutively active MAPK kinase 1 (MEK1) mutant or by myristoylated Akt. In addition, suppression of p38 MAPK activation by overexpression of a dominant negative p38 MAPK or MKK6 mutant did not diminish insulin-induced glucose uptake by 3T3-L1 adipocytes. It is thus apparent that activation of p38 MAPK is not essential for insulin-induced increases in glucose uptake. Rather, p38 MAPK activation leads to a marked down-regulation of insulin-induced glucose uptake via GLUT4, which may underlie cellular stress-induced insulin resistance caused by tumor necrosis factor alpha and other factors.

3T3 Cells↗
PubMed2001

Five isoforms of the phosphatidylinositol 3-kinase regulatory subunit exhibit different associations with receptor tyrosine kinases and their tyrosine phosphorylations.

There are five isoforms of the regulatory subunit for the heterodimeric type of phosphatidylinositol 3-kinase. These five regulatory subunit isoforms were overexpressed using an adenovirus transfection system, and their own tyrosine phosphorylations and associations with various tyrosine kinase receptors were investigated. When overexpressed in CHO-PDGFR cells, the associations of these regulatory subunit isoforms with the platelet-derived growth factor receptor were similar. However, when overexpressed in CHO-IR cells, p55gamma exhibited a significantly lower ability to bind with IRS-1 upon insulin stimulation, as compared with other regulatory subunit isoforms. Furthermore, p55alpha and p55gamma were found to be tyrosine-phosphorylated. Finally, interestingly, when overexpressed in CHO-EGFR cells or A431 cells and stimulated with epidermal growth factor (EGF), phosphorylated EGF receptor was detected in p85alpha, p85beta and p50alpha immunoprecipitates, but not in p55alpha and p55gamma immunoprecipitates. In addition, EGF-induced tyrosine phosphorylation was observed in p85alpha, p85beta, p55alpha and p55gamma, but not in p50alpha, immunoprecipitates. Thus, each regulatory subunit exhibits specific responses regarding both the association with tyrosine-phosphorylated substrates and its own tyrosine phosphorylation. These results suggest that each isoform possesses specific roles in signal transduction, based on its individual tyrosine kinase receptor.

Adenoviridae↗
PubMed2001

G649, an allelic variant of the human H2 receptor with low basal activity, is resistant to upregulation upon antagonist exposure.

Orange et al reported an allelic variant of the human histamine H2 receptor, in which adenine 649 was replaced with guanine, to be more frequent in the schizophrenic population than controls in British Caucasians. The A649 to G change causes an Asn to Asp transition at amino acid position 217 in the third intracellular region, which is postulated to be important for receptor function. Herein, we analyzed the functional significance of this variant using wild-type and variant receptors expressed in Chinese hamster ovary cells. The variant receptor was associated with markedly lower basal cAMP productions than the wild-type receptor. Histamine-dependent cAMP productions via the variant receptor were lower as well. Treatment of cells expressing variant receptors with 10(-5) M ranitidine for 24 h resulted in a reduced degree of receptor upregulation as compared with the wild-type receptor. Thus, this is the first report of an allelic variant of the human H2 receptor which confers altered receptor function. To analyze gastric acid secretion in individuals with this variant, we examined 100 Japanese control subjects. However, neither heterozygotes nor homozygotes were found, suggesting that this variant, if present, is uncommon in the Japanese population.

Alleles↗
PubMed2001

Endoscopically demonstrable esophageal changes after Helicobacter pylori eradication in patients with gastric disease.

BACKGROUND AND AIMS: An increased prevalence of reflux esophagitis has been reported following Helicobacter pylori (H. pylori) eradication in patients with duodenal ulcers in Western countries. However, it has remained unknown whether this might also appertain to individuals with other diseases. We therefore carried out this study to determine the effect of eradicating H. pylori infection in a series of Japanese patients. METHODS: Of a total of 203 H. pylori-positive patients successfully cured of infection, 82 cases (58 males, 24 females) with gastric disease, but not duodenal ulcers, were included in the present study; median age 56 years (range 18-80) and median follow up of 24 months (range 6-65). The patients were investigated clinically and endoscopically at regular intervals. RESULTS: Mild reflux esophagitis developed after eradication in three of 55 (5.5%) patients formerly without this condition, while it improved after eradication in five of 27 (18.5%) patients, with the disease endoscopically diagnosed prior to eradication. The estimated incidence of esophagitis within 3 years was 4.8% after cure of infection. Short segment Barrett's esophagus developed after eradication in six of 58 (10.3%) patients who did not have it prior to the therapy, while the condition did not improve in 24 patients affected before eradication. CONCLUSIONS: Endoscopic esophageal changes after H. pylori eradication in the present series of Japanese patients were relatively infrequent and mild. This therapeutic approach thus appears to be safe and unproblematic.

Adolescent↗
PubMed2001

Detection of t(I 1; 18) in MALT-type lymphoma with dual-color fluorescence in situ hybridization and reverse transcriptase-polymerase chain reaction analysis.

t(11:8) is a recurrent chromosomal abnormality observed in mucosa-associated lymphoid tissue (MALT)-type lymphoma. API2 and MLT genes have been implicated. The authors devised a dual-color interphase fluorescence in situ hybridization (FISH) system to detect splitting of 11q22 and its fusion with 18q21. Subjects were 44 cases of extranodal lymphoma and cases of primary macroglobulinemia. Whenever RNA was available, reverse transcriptase-polymerase chain reaction followed by sequence analysis was performed. Positive cases by dual-color FISH analysis were restricted to MALT-type lymphoma and one case of primary macroglobulinemia. Among 24 cases of MALT-type lymphoma, 14 (58%) (4 gastric, 5 pulmonary, 3 orbital, 1 salivary, and 1 thyroid lymphomas) had splitting of the 11q22 region probes and fusion of signals suggesting the translocation of chromosome 11 and 18. Reverse transcriptase-polymerase chain reaction analysis showed the API2/MLT gene fusion in 9 of 10 cases. Sequence analyses showed three different modes of involvement of the MLT gene, whereas the breakpoint at API2 was the same. Monoclonal component of serum immunoglobulin M was observed in 3 of 14 positive cases for the translocation. Direct visualization using dual-color FISH on samples serves as a molecular tool for management of MALT-type lymphoma with API2/MLT gene fusion.

Adult↗
PubMed2001

Regulation of phosphoinositide metabolism, Akt phosphorylation, and glucose transport by PTEN (phosphatase and tensin homolog deleted on chromosome 10) in 3T3-L1 adipocytes.

To investigate the roles of PTEN (phosphatase and tensin homolog deleted on chromosome 10) in the regulation of 3-position phosphorylated phosphoinositide metabolism as well as insulin-induced Akt phosphorylation and glucose metabolism, wild-type PTEN and its phosphatase-dead mutant (C124S) with or without an N-terminal myristoylation tag were overexpressed in Sf-9 cells and 3T3-L1 adipocytes using baculovirus and adenovirus systems, respectively. When expressed in Sf-9 cells together with the p110alpha catalytic subunit of phosphoinositide 3-kinase, myristoylated PTEN markedly reduced the accumulations of both phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate induced by p110alpha. In contrast, overexpression of the C124S mutants apparently increased these accumulations. In 3T3-L1 adipocytes, insulin-induced accumulations of phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate were markedly suppressed by overexpression of wild-type PTEN with the N-terminal myristoylation tag, but not by that without the tag. On the contrary, the C124S mutants of PTEN enhanced insulin-induced accumulations of phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate. Interestingly, the phosphorylation level of Akt at Thr308 (Akt2 at Thr309), but not at Ser473 (Akt2 at Ser474), was revealed to correlate well with the accumulation of phosphatidylinositol 3,4,5-trisphosphate modified by overexpression of these PTEN proteins. Finally, insulin-induced increases in glucose transport activity were significantly inhibited by the overexpression of myristoylated wild-type PTEN, but were not enhanced by expression of the C124S mutant of PTEN. Therefore, in conclusion, 1) PTEN dephosphorylates both phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate in vivo, and the C124S mutants interrupt endogenous PTEN activity in a dominant-negative manner. 2) The membrane targeting process of PTEN may be important for exerting its function. 3) Phosphorylations of Thr309 and Ser474 of Akt2 are regulated differently, and the former is regulated very sensitively by the function of PTEN. 4) The phosphorylation level of Ser474, but not that of Thr309, in Akt2 correlates well with insulin-stimulated glucose transport activity in 3T3-L1 adipocytes. 5) The activity of endogenous PTEN may not play a major role in the regulation of glucose transport activity in 3T3-L1 adipocytes.

3T3 Cells↗
PubMed2001

Insulin resistance with enhanced insulin signaling in high-salt diet-fed rats.

Previous clinical studies showed an apparent correlation between hypertension and insulin resistance, and patients with diabetes are known to have increased blood pressure responsiveness to salt loading. To investigate the effect of high salt intake on insulin sensitivity and the insulin signaling pathway, a high-salt diet (8% NaCl) or a normal diet was given to 7-week-old SD rats for 2 weeks. High salt-fed rats developed slightly but significantly higher systolic blood pressure than controls (133 +/- 2 vs. 117 +/- 2 mmHg, P < 0.001), with no change in food intake or body weight. High salt-fed rats were slightly hyperglycemic (108.5 +/- 2.8 vs. 97.8 +/- 2.5 mg/dl, P = 0.01) and slightly hyperinsulinemic (0.86 +/- 0.07 vs. 0.61 +/- 0.06 ng/ml, P = 0.026) in the fasting condition, as compared with controls. Hyperinsulinemic-euglycemic clamp study revealed a 52.7% decrease in the glucose infusion rate and a 196% increase in hepatic glucose production in high salt-fed rats, which also showed a 66.4% decrease in 2-deoxyglucose uptake into isolated skeletal muscle and a 44.5% decrease in insulin-induced glycogen synthase activation in liver, as compared with controls. Interestingly, despite the presence of insulin resistance, high salt-fed rats showed enhanced insulin-induced tyrosine phosphorylation of insulin receptor substrate (IRS)-1, IRS-2 (liver and muscle), and IRS-3 (liver only). Phosphatidylinositol (PI) 3-kinase activities associated with IRS and phosphotyrosine in the insulin-stimulated condition increased 2.1- to 4.1-fold, as compared with controls. Insulin-induced phosphorylation of Ser-473 of Akt and Ser-21 of glycogen synthase kinase-3 also increased 2.9- and 2-fold, respectively, in the liver of the high salt-fed rats. Therefore, in both the liver and muscle of high salt-fed rats, intracellular insulin signaling leading to PI 3-kinase activation is enhanced and insulin action is attenuated. The hyperinsulinemic-euglycemic clamp study showed that decreased insulin sensitivity induced with a high-salt diet was not reversed by administration of pioglitazone. The following can be concluded: 1) a high-salt diet may be a factor promoting insulin resistance, 2) the insulin-signaling step impaired by high salt intake is likely to be downstream from PI 3-kinase or Akt activation, and 3) this unique insulin resistance mechanism may contribute to the development of diabetes in patients with hypertension.

Animals↗
PubMed2000

Phase I study of a weekly infusion of irinotecan hydrochloride (CPT-11) and a 14-day continuous infusion of etoposide in patients with lung cancer: JCOG trial 9408.

BACKGROUND: The aim was to determine the maximum tolerated dose (MTD) and recommended dose of irinotecan hydrochloride (CPT-11) in combination with a 14-day continuous infusion of etoposide in patients with refractory advanced lung cancer (LC), especially small cell lung cancer (SCLC). METHODS: Etoposide was administered continuously at 25 mg/m2/day for 14 days. The initial dose of CPT-11 was 40 mg/m2 given as a 90 min intravenous infusion on days 1, 8 and 15 and the dose escalation of CPT-11 was planned in increments of 20 mg/m2 until severe or life-threatening toxic effects were observed. RESULTS: Nine refractory or advanced LC patients (eight at level 1, one at level 2) were entered in this study, of whom two at level 1 were not assessable for toxicity because of patient's refusal and progressive disease. One treatment-related death due to pulmonary toxicity and one patient with hypotension who needed catecholamine for more than 48 h were observed at level 1, a CPT-11 dose of 40 mg/m2. The MTD of CPT-11 was 40 mg/m2. Therapeutic efficacy could be assessed in seven patients, of whom two achieved a partial response. CONCLUSIONS: This regimen was too toxic and the recommended dose was outside the levels in this study. One has to consider pulmonary toxicity when using CPT-11, especially for patients previously treated with cytotoxic agents for which pulmonary toxicity has been reported.

Adenocarcinoma↗
PubMed2000

Dexamethasone-induced insulin resistance in 3T3-L1 adipocytes is due to inhibition of glucose transport rather than insulin signal transduction.

Glucocorticoids reportedly induce insulin resistance. In this study, we investigated the mechanism of glucocorticoid-induced insulin resistance using 3T3-L1 adipocytes in which treatment with dexamethasone has been shown to impair the insulin-induced increase in glucose uptake. In 3T3-L1 adipocytes treated with dexamethasone, the GLUT1 protein expression level was decreased by 30%, which possibly caused decreased basal glucose uptake. On the other hand, dexamethasone treatment did not alter the amount of GLUT4 protein in total cell lysates but decreased the insulin-stimulated GLUT4 translocation to the plasma membrane, which possibly caused decreased insulin-stimulated glucose uptake. Dexamethasone did not alter tyrosine phosphorylation of insulin receptors, and it significantly decreased protein expression and tyrosine phosphorylation of insulin receptor substrate (IRS)-1. Interestingly, however, protein expression and tyrosine phosphorylation of IRS-2 were increased. To investigate whether the reduced IRS-1 content is involved in insulin resistance, IRS-1 was overexpressed in dexamethasone-treated 3T3-L1 adipocytes using an adenovirus transfection system. Despite protein expression and phosphorylation levels of IRS-1 being normalized, insulin-induced 2-deoxy-D-[3H]glucose uptake impaired by dexamethasone showed no significant improvement. Subsequently, we examined the effect of dexamethasone on the glucose uptake increase induced by overexpression of GLUT2-tagged p110alpha, constitutively active Akt (myristoylated Akt), oxidative stress (30 mU glucose oxidase for 2 h), 2 mmol/l 5-aminoimidazole-4-carboxamide ribonucleoside for 30 min, and osmotic shock (600 mmol/l sorbitol for 30 min). Dexamethasone treatment clearly inhibited the increases in glucose uptake produced by these agents. Thus, in conclusion, the GLUT1 decrease may be involved in the dexamethasone-induced decrease in basal glucose transport activity, and the mechanism of dexamethasone-induced insulin resistance in glucose transport activity (rather than the inhibition of phosphatidylinositol 3-kinase activation resulting from a decreased IRS-1 content) is likely to underlie impaired glucose transporter regulation.

3T3 Cells↗
PubMed1999

Efficient coupled transcription/translation from PCR template by a hollow-fiber membrane bioreactor.

A novel bioreactor using a hollow-fiber membrane was developed for the coupled transcription/translation system using T7 RNA polymerase and Escherichia coli S30 extract. The large surface area per the reaction volume of the reactor assured rapid mass transfers of substrates into the reaction mixture and of wastes out from it across the membrane by their molecular diffusion. The flux was large enough to maintain nucleotide concentrations for more than 3 h, which increased the protein synthesis greatly. In addition, the T7 terminator sequence downstream from the reporter genes was found to increase the synthesized protein significantly, especially when the product of polymerase chain reaction (PCR) was used as a template. Implementation of this finding and use of the bioreactor developed multiplied the productivity of protein by the in vitro direct expression from PCR template.

Base Sequence↗
PubMed1999

[A case of fulminant Mycoplasma pneumoniae pneumonia successfully treated by steroid therapy].

A 35-year-old previously healthy woman was admitted because of pulmonary infiltrates in both lungs and respiratory failure associated with Mycoplasma pneumoniae infection. Although she initially required temporary mechanical ventilatory assistance, her clinical condition improved dramatically in response to steroid therapy. In this report, we discussed the effectiveness of steroid therapy in treating a case of severe M. pneumoniae pneumonia.

Adult↗
PubMed1999

Endoscopic and histological reversibility of gastric adenoma after eradication of Helicobacter pylori.

Although epidemiological studies strongly suggest an association between Helicobacter pylori infection and gastric carcinogenesis via a multistage process, a causal link between them has not been demonstrated. We evaluated the endoscopic and histological changes of gastric adenoma, which is considered a premalignant condition, after eradication of H. pylori. Thirty-five H. pylori-infected patients with gastric adenoma were treated with triple therapy (lansoprazole 30mg/day, clarithromycin 400mg/day, and amoxicillin 1500 mg/day) for 1 week. Of these 35 patients, 30 (86%) exhibited no H. pylori by culture or histology after the therapy. Of the 30 gastric adenomas, 7 decreased in size endoscopically; three gastric adenomas especially showed apparent remission, although histological cure in these three patients was not apparent. Our results suggest that removal of H. pylori infection may only mask a gastric adenoma endoscopically owing to the change around the gastric mucosa.

2-Pyridinylmethylsulfinylbenzimidazoles↗
PubMed1998

[Anterior ischemic optic neuropathy in a case of polyarteritis nodosa].

A 68-year-old male admitted to our hospital because of fever, body weight loss and multiple mononeuropathy. He developed visual loss in his right eye due to anterior ischemic optic neuropathy (AION). Surgical treatment of jejunal perforation was performed and histological examination of jejunal ulcer confirmed the diagnosis of polyarteritis nodosa (PN). Combination therapy of steroid (prednisolone 50 mg/day) and cyclophosphamide (50 mg/day) was started, which saved him from any other ischemic change including that of his left eye. As long as we know, AION occurring in PN is very rare, that is, only 4 cases have been reported in Japan.

Aged↗